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Use of a scaffold peptide in the biosynthesis of amino acid–derived natural products

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Science  19 Jul 2019:
Vol. 365, Issue 6450, pp. 280-284
DOI: 10.1126/science.aau6232

Growing independently

Bacteria produce natural products with a range of functions. A major family comprises ribosomally synthesized and posttranslationally modified peptides. Ting et al. identify a biosynthetic pathway in which a natural product is derived from an amino acid that is added to a ribosomally synthesized peptide independent of the ribosome. This biosynthetic paradigm is used in the synthesis of thiaglutamate and ammosamides, and the finding of related gene clusters suggests that the strategy may be used more widely.

Science, this issue p. 280

Abstract

Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA–dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid–derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid–derived natural products.

Bacteria produce many small-molecule natural products that play important roles in communication, symbiosis, and competition (1). Historically, these compounds have been discovered by activity-based screens. An alternative avenue for their discovery starts with identification of their biosynthetic gene clusters, now that bacterial genomes have revealed the tremendous diversity of natural products that remain to be discovered (2). In this study we focus on a group of biosynthetic gene clusters for which the final products were not known and could not be predicted.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) (3) include lantibiotics and thiopeptides that are used in food and agriculture (4). They are biosynthesized from a precursor peptide consisting of a leader peptide that serves as a recognition motif for the biosynthetic enzymes and a core peptide that is converted to the final product. During their maturation, Ser and Thr residues are glutamylated by LanB enzymes through a glutamyl-tRNA–dependent mechanism (5, 6). Subsequently, the glutamate is eliminated to generate dehydroamino acids (Fig. 1A). A survey of >100,000 publicly available bacterial genomes revealed more than 600 genes that encode LanB-like proteins in which the elimination domain is not present within the cluster or genome.

Fig. 1 Function of a small LanB enzyme, TglB, found in P. syringae.

(A) LanB enzymes glutamylate Ser/Thr residues and subsequently eliminate the glutamate to form dehydroamino acids. Small LanB proteins lack the elimination domain. Dha, dehydroalanine; Dhb, dehydrobutyrine. (B) Biosynthetic gene cluster in P. syringae that encodes a small LanB (TglB). (C) Matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectra of TglA coexpressed with TglB. (D) Analysis of the TglB product by electrospray ionization–tandem MS (ESI-MS/MS).

In the plant pathogen Pseudomonas syringae pv. maculicola ES4326, such a protein (TglB) is encoded near an open reading frame for a 50-amino-acid peptide (TglA) (Fig. 1B). Coexpression of His6-TglA and -TglB in Escherichia coli and subsequent purification of the peptide demonstrated an increase in mass by 103 Da (Fig. 1C). This increase is inconsistent with glutamylation but could be the result of condensation with a cysteine residue. High-resolution tandem mass spectrometry (MS/MS) analysis of the peptide suggested that the adduct was attached to the C-terminal alanine instead of the anticipated ester linkage to a Ser in the peptide (Fig. 1D). We expressed TglA and TglB individually as His6-tagged proteins and purified them. In vitro incubation with Cys, ATP, tRNACys, and Cys tRNA synthetase (CysRS) resulted in the same product (TglA-Cys) (Fig. 2A) as that isolated from co-expression in E. coli, confirming that TglB adds a Cys to the C terminus of TglA in a tRNA-dependent manner (fig. S1A). This C-terminal peptide extension not only constitutes a previously unknown posttranslational modification but also seems counterintuitive, because a more logical route to the product would entail a Cys encoded by tglA. We next purified Cys-tRNACys and showed that TglB does not transfer the Cys to the C terminus of TglA unless ATP is present, which is converted to ADP and phosphate (fig. S1B). Performing the reaction in buffer made with H218O and subsequent MS analysis demonstrated that the product contains one 18O atom (fig. S1C), and addition of hydroxyl amine to the assay mixture allowed trapping of C-terminally activated TglA as the hydroxamate (fig. S1D). These findings are consistent with activation of the C terminus of TglA by phosphorylation, subsequent amide bond formation with the amino group of Cys-tRNA, and release of the tRNA by hydrolysis (fig. S1E, mechanism 1). The observations rule out the use of the activated ester of Cys-tRNA for the nonribosomal peptide extension (fig. S1E, mechanism 2). TglB accepted a 12-mer peptide corresponding to the C terminus of TglA as a minimal substrate (fig. S1F), and kinetic experiments showed that TglB has a turnover of 28 min−1 in the presence of full-length TglA (fig. S1G).

Fig. 2 The cysteine added by TglB is modified by other enzymes encoded by the tgl cluster.

(A) Inferred biosynthetic pathway toward 3-thiaglutamate. (B) MALDI-TOF mass spectrum of in vitro reaction of TglHI with TglA-Cys. (C) MALDI-TOF mass spectrum of in vitro reaction of TglF with compound 1. Color coding of shaded peaks in (B) and (C) is shown in (A).

We next interrogated the other proteins encoded in the biosynthetic gene cluster. TglH has low homology to a structurally characterized dinuclear nonheme iron–dependent protein for which no activity has been reported (7). The C-terminal domain of TglI has homology with known leader peptide-binding domains in RiPP biosynthetic enzymes (Fig. 1B) (5, 8). We co-expressed TglA with TglB, TglH, and TglI in E. coli and isolated a product that was decreased in mass by 14 Da from TglA-Cys (fig. S2A). We treated the peptide with trypsin to generate a C-terminal tetrapeptide. Chemical assays with thiol- and carboxylate-reactive electrophiles indicated that the product still contained these functional groups (fig. S3), suggesting structure 1 as the product of TglHI (Fig. 2A). We next repeated this experiment but used an E. coli strain that is auxotrophic for Cys and that was grown in minimal medium supplemented with 13C-labeled Cys. Isolation of the peptide and analysis by MS showed that it is the cysteine β carbon that is removed (fig. S4).

The biosynthetic cluster also contains a pair of genes (tglEF) encoding proteins similar to a recently characterized carboxy-S-adenosylmethionine (Cx-SAM) synthase and a Cx-SAM–dependent methyltransferase, respectively (9, 10). We added compound 1 to Cx-SAM and TglF in vitro and isolated product 2, with a mass increase of 58 Da (Fig. 2C), consistent with carboxymethylation of a thiol. This hypothesis was confirmed by treating the TglHI product with iodoacetic acid, which resulted in the same outcome, as did coexpression of TglABEFHI in E. coli (fig. S2B). The in vitro–prepared peptide was treated with trypsin and the C-terminal tetrapeptide 3 was characterized by 1H nuclear magnetic resonance (NMR) spectroscopy and tandem MS, which supported structure 2 for the TglF product (Fig. 2A and fig. S5). Given the unusual architecture, we also chemically synthesized peptide 3 as two diastereomers (supplementary materials and methods) and demonstrated that the 1H NMR spectrum of one isomer was identical to that of the enzymatic product (fig. S5). We tried to obtain crystals to assign the stereochemistry of either isomer and made several chemical derivatives but were unable to obtain crystals for X-ray diffraction.

We next turned to the cryo–electron microscopy (cryo-EM) method microcrystal electron diffraction (MicroED) (1113). A small amount of powder of the diastereomer that eluted first during high-performance liquid chromatography purification was placed onto an EM grid, plunged into liquid nitrogen, and investigated under cryogenic conditions in an electron microscope. The seemingly amorphous powder contained numerous nanocrystals on the grid that were suitable for MicroED analysis, each consisting roughly of femtograms of material that diffracted to ~1-Å resolution. MicroED data were collected from each nanocrystal, but the sample was highly susceptible to beam damage such that no useful diffraction was observed after the first few frames of the MicroED movie. Despite collection of >150 datasets on a complementary metal oxide semiconductor-based CetaD camera, nanocrystals succumbed to radiation damage too fast, preventing structure determination. It is possible that the peptide was particularly susceptible to damage because of the 3-thiaglutamate, consistent with an earlier study that showed that radiation damage is particularly prevalent at Cys residues (14). We then turned to the Falcon III direct electron detector, one of the most sensitive cameras for cryo-EM that was recently demonstrated to be suitable for MicroED data collection and structure determination and that minimizes radiation damage because of its high sensitivity and high frame rate (15). Atomic resolution data from seven nanocrystals were collected, each covering an angular range of ~50° before damage was observed. Data from five nanocrystals were merged to yield a 96% complete dataset to 1.0-Å resolution, and the structure was determined by direct methods (Fig. 3; PDB 6PO6; EMD-20411 crystallographic data in table S3 and supplementary materials and methods). The atomic resolution MicroED structure revealed the d configuration of the 3-thiaGlu in this peptide (D-3), which in turn provided the stereochemical assignment for L-3, which coelutes with and has the same spectral data as the enzymatic product. These results demonstrate that the TglHI-catalyzed reaction occurred with retention of configuration at the α carbon (Fig. 3, B to D). These findings highlight the utility of MicroED to determine the structure and stereochemistry of a previously unknown natural product. Thus, collectively, TglBEFHI convert TglA into a peptide containing L-3-thiaglutamate at its C terminus (TglA-thiaGlu; 2 in Fig. 2A).

Fig. 3 In vitro TglHI reacts with 13C-labeled TglA-Cys to produce 13C-formate and compound 1 with retention of configuration.

(A) Diffraction pattern of D-3 with resolution ring at 0.9 Å. (B) Atomic MicroED structure of D-3 determined at 1.0-Å resolution. (C) Structure of chemically synthesized tetrapeptides (VFA-thiaGlu) containing D-thiaGlu (D-3) and L-thiaGlu (L-3). (D) Determination of stereochemical configuration of thiaGlu by comparison with synthetic standards. High-performance liquid chromatograms are shown. VFA-thiaGlu was obtained by TglHI modification of TglA-Cys and then 2-iodoacetic acid alkylation and trypsin digest. (E) 13C NMR spectra showing the β carbon of the C-terminal cysteine in 13C-labeled TglA-Cys (26.3 ppm; top), and a new signal at 171.0 ppm that corresponds to 13C-formate after reaction with TglHI (bottom).

We next investigated the TglHI-catalyzed reaction with purified proteins. Neither protein could be expressed in soluble form individually, but coexpression resulted in copurification and metal analysis indicated TglHI contained 2.5 Fe. In vitro TglHI converted TglA-Cys to 1 under aerobic conditions with a turnover of 1.1 min−1 (Fig. 2B), whereas under low oxygen concentrations product formation was negligible, confirming oxygen dependency of the reaction (fig. S6A). To investigate if TglHI can functionalize internal cysteine residues, the extension mutant TglA-CysAla was prepared. This peptide was not modified by TglHI (fig. S6B). TglHI also did not modify other unrelated peptides that end in Cys (fig. S6C), and N-terminal truncation of TglA-Cys led to diminished or abolished TglHI activity (fig. S6D). Thus, the enzyme has high specificity for TglA-Cys. To identify the fate of the lost carbon atom, 13C-labeled TglA-Cys was reacted with TglHI, and formate was observed by 13C NMR spectroscopy (Fig. 3E). Moreover, when [2,3,3,2H3]Cys was used, the product contained one deuterium, illustrating that the α hydrogen is likely not removed during the transformation (fig. S4D). Thus, TglHI catalyzes a net four-electron oxidation of TglA-Cys, modifying the redox states of both the α and β carbons of the C-terminal cysteine installed by TglB. Based on the in vitro studies, we propose a mechanism for the formation of 1 and formate from TglA-Cys (fig. S7). The chemistry catalyzed by TglHI expands the range of posttranslational modifications in natural product biosynthesis (16) to include a remarkable excision of a methylene group from cysteine. Additional TglHI-like enzymes are encoded in the genomes (fig. S8), including in the biosynthetic gene cluster for methanobactin (1719).

The last four genes in the biosynthetic cluster encode a putative membrane-bound protease (TglG), a putative pyridoxal-phosphate–dependent enzyme (TglC) that is sometimes missing in homologous clusters, and two putative transporters (TglD and TglJ). Like TglB and TglI, TglG contains a RiPP leader peptide recognition motif, suggesting that it will act on a TglA-derived peptide (Fig. 1B), and homologous enzymes have cytoplasmic active sites (20). When TglA-thiaGlu was exposed to the membrane fraction of cell lysate of E. coli expressing green fluorescent protein–tagged TglG, the peptide was cleanly converted into TglA (fig. S9). TglA-Glu was also a substrate, but TglA-GluAla was not, illustrating that the protease cannot distinguish Glu and 3-thiaGlu but does not tolerate extension of the peptide. Thus, TglA appears to be a scaffold on which 3-thiaGlu is assembled and final proteolytic release regenerates TglA for another round of biosynthesis (Fig. 2A). Were cysteine merely encoded by tglA, then each ribosomally produced peptide could make only a single 3-thiaglutamate. Instead, the use of TglA as a scaffold peptide is conceptually more efficient than the stoichiometric use of leader peptide in other RiPP pathways (4). At present we do not know the function of 3-thiaGlu, nor whether this unstable compound is further chemically modified. Plants were recently shown to use Glu for a systemic signaling response to pathogens (21), and it is possible that 3-thiaGlu or a product derived from it interferes with Glu signaling similarly to other antimetabolite toxins made by P. syringae that block jasmonate and ethylene signaling pathways (22).

We note that 3-thiaGlu is not a RiPP, because it is not ribosomally synthesized, but it is made by posttranslational modification reactions. Perhaps this unusual pathway evolved because of the significant relative burden of leader peptide production for a single amino acid product. Bioinformatic prediction of TglA transcriptional regulation (23) suggests that precursor production is not driven by a separate promoter, which is consistent with putative catalytic use of the peptide (fig. S10). This contrasts with most RiPP pathways in which expression of the substrate peptide is controlled by its own promoter followed by a readthrough transcriptional terminator to allow the precursor peptide to be present in excess over the biosynthetic machinery (24, 25).

It is the Cys-tRNA–dependent enzyme TglB that allows the proposed catalytic use of TglA. Similar small LanB-encoding genes are found in several bacterial phyla, with some clusters encoding multiple such proteins and a range of additional putative modification enzymes (fig. S11). To assess the generality of the function of small LanB proteins and provide further support for a catalytic role of the scaffold peptide, we investigated ammosamide biosynthesis. A previous study of these Trp-derived pyrroloquinoline natural products (Fig. 4A) hinted that the compounds could be derived from a small peptide, AmmA ending in Trp, encoded in the gene cluster (Fig. 4B) (26). However, when this Trp was mutated to Ser or deleted altogether, ammosamide was still produced (26). The ammosamide gene cluster encodes four small LanB proteins. We tested all four for activity in vitro and in E. coli with AmmA (previously annotated Amm6) and AmmA lacking the C-terminal Trp, but we observed no activity. We noted that AmmA has homology with other peptides encoded in clusters with small LanB proteins (Fig. 4B) but that AmmA appears to have a C-terminal extension. When we removed this extension, AmmB2 (previously annotated Amm9), but not the other three AmmB proteins, added a Trp in a Trp-tRNA–dependent fashion to the C terminus of the peptide in vitro and in E. coli (Fig. 4C). This finding explains the observation that mutation or deletion of the C-terminal Trp still resulted in ammosamide production and supports catalytic use of the peptide. Such use provides an attractive explanation for the 134 mg/L of ammosamide C generated by the producing bacterium (26), because stoichiometric use would require production of 3.0 g of AmmA. Given this second example of tRNA-dependent activity, we suggest the name peptide-amino acyl tRNA ligase (PEARL) for the small LanB proteins. The biosynthesis of a metabolite on a small peptide scaffold is uncommon, with the closest similarity found in the biosynthesis of amino acids linked by isopeptide bonds to a glutamate residue on amino-carrier proteins in some bacteria (27, 28).

Fig. 4 Ammosamide biosynthesis involves addition of L-Trp to the C terminus of a ribosomally synthesized peptide.

(A) Pyrolloquinoline alkaloid ammosamides A to C. (B) Sequence alignment of the C terminus of the AmmA precursor peptide and its homologs, showing a C-terminal extension for AmmA relative to most homologs. The gene cluster for ammosamide biosynthesis in Streptomyces sp. CNR698 comprises 27 open reading frames. The encoded proteins include four small LanBs, two proteases, one halogenase, and a transporter. (C) AmmB2 adds L-Trp to AmmA*, a truncated peptide of AmmA, to afford AmmA*W in vitro in an ATP–, tRNATrp–, and Trp-RS–dependent reaction. The red MALDI-TOF mass spectrum is AmmA*, and the blue spectrum shows the product of the reaction. High-resolution ESI-MS/MS confirmed addition of L-Trp to the C terminus.

Supplementary Materials

science.sciencemag.org/content/365/6450/280/suppl/DC1

Materials and Methods

Scheme 1

Figs. S1 to S11

Tables S1 to S3

References (2949)

References and Notes

Acknowledgments: P. syringae pv. maculicola ES4326 was provided by D. Desveaux (University of Toronto). We thank R. Splain (van der Donk lab) and M. W. Martynowycz and J. Hattne (Gonen lab, UCLA) for advice and useful discussions. Funding: This work was supported by the National Institutes of Health (R37 GM058822 to W.A.v.d.D.; F32 GM129944 to C.P.T.; F32 GM120868 to M.A.F.). T.G. and W.A.v.d.D. are investigators of the Howard Hughes Medical Institute. Author contributions: C.P.T., M.A.F., and Z.Z. performed biochemical assays. M.A.F. performed bioinformatics analysis. C.P.T., M.A.F., and W.A.v.d.D. designed experiments, analyzed data, and wrote the manuscript. S.L.H and T.G. designed the MicroED experiment; performed MicroED data collection, processing, and refinement of the structure; and contributed to writing of the manuscript and figure preparation. Competing interests: The authors declare no competing financial interests. M.A.F. is employed by the American Association for the Advancement of Science, and his editorial access to the paper was blocked. Data and materials availability: All data supporting the findings in this study are provided in the main text and supplementary materials.

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